doi: 10.1242/10.1242/jcs.00042
Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes
Kirsi Rilla1,
Mikko J. Lammi1,
Reijo Sironen1,
Kari Törrönen1,
Merja Luukkonen1,
Vincent C. Hascall2,
Ronald J. Midura2,
Mika Hyttinen1,
Jukka Pelkonen3,
Markku Tammi1 and
Raija Tammi1,*
1 Department of Anatomy, University of Kuopio, 70211 Kuopio, Finland
2 Department of Biomedical Engineering, Lerner Research Institute, Cleveland
Clinic Foundation, Cleveland, Ohio 44195, USA
3 Department of Clinical Microbiology, University of Kuopio, 70211 Kuopio,
Finland
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Fig. 1. Total Has2 mRNA levels in REK clones transfected with Has2 sense and
antisense plasmids. RNA from three to four separate RNA isolations from each
antisense (A9-A25) and sense (S25-S30) cell line was subjected to RT-PCR
analysis. The electrophoretic band densities relative to GAPDH were compared
to those with vector only (mock)-transfected cell lines within each
experiment. The results are expressed as a percentage of mock controls
(±s.e.) for each individual cell line.
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Fig. 2. Hyaluronan production in Has2-transfected cells. (A) Nearly confluent
cultures were stained for cell-associated hyaluronan using the bHABC probe.
Wt, wildtype; M2, mock; A22, antisense; S29, sense cell line. Note the
elongation of some of the most intensely stained cells in the S29 sense line
(arrows). (B) Optical densities of the DAB signal, representing
cell-associated hyaluronan, were assayed in cultures of equal density to those
in (A) using microscopic image analysis. The bars show the s.e. of 10 randomly
taken microscopic fields. (C) Hyaluronan secreted into the growth medium by
the sense (S25-S30), antisense (A9-A25) and mock-transfected (M2) cells was
determined as fluorophore-tagged, enzymatically liberated disaccharides and
plotted as a function of cell density. Linear regression lines for the
antisense and sense cell lines are shown.
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Fig. 3. Spreading of the transfected cell lines. (A) Differential interference
contrast micrographs of typical mock- (M2) and antisense- (AS22) transfected
cells 24 hours after plating. The small lamellipodia in antisense cells are
depicted by arrows. (B) Substratum areas occupied by individual cells 6 hours
after plating. The data represent means±s.e. of three independent
experiments. One-way analysis of variance showed that the cell lines were
significantly different (P< 0.01). The cell lines indicated by an
asterisk significantly differ from all of the mock lines (Fisher test,
P<0.05). (C) The time course of the spreading of the transfected
REKs. The areas of individual cells were measured at different time points
after plating. The points indicate means±s.e. of 21-96 randomly taken
cells for each time and cell line. W, wildtype; M1-M3, mock controls; S25-S30,
sense cells; A9-A25, antisense cells.
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Fig. 4. Vinculin-positive adhesion plaques and hyaluronan at the cell-substratum
interface. (A) Double immunofluorescence for hyaluronan (green) and vinculin
(red) in confocal optical sections constituting the undersurface of the
keratinocytes. The image of vinculin is processed as for the quantification of
adhesion plaques, see Materials and Methods. (B) Quantification of
vinculin-positive areas by image analysis in the transfected cell lines. Means
and ranges of two separate experiments for each cell line are shown. One-way
analysis of variance and Fisher tests were used to indicate the cell lines
that were significantly different *(P<0.05) from mock
controls.
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Fig. 5. Proliferation rate of Has2-transfected cell lines. Cell numbers were
counted with a hemocytometer at different time points after plating an equal
number of cells from each cell line. (A) Has2 antisense cell lines (A22, A23,
A25) with wild-type (Wt) and mock-transfection (M2) controls. (B) Has2 sense
cells (S27, S29, S30) and the controls. Bars represent the range of duplicate
cultures. The antisense lines differed significantly from all other cell types
on day 1 (*P<0.05, analysis of variance).
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Fig. 6. Migration of the Has2-transfected cell lines following wounding of cell
monolayers. A cell-free area was introduced by scraping each monolayer
crosswise with a sterile pipette tip. The area covered by the cells was
measured immediately after scraping and 24 hours later. The data represent
means±s.e. from six separate experiments. One-way analysis of variance
showed that the cell lines indicated by an asterisk were significantly
different from the mock lines (P<0.05).
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Fig. 7. Induction of hyaluronan synthesis in the wound edge. Confluent cultures
were treated with Streptomyces hyaluronidase to remove existing
hyaluronan, scraped with a pipette tip, washed and 8 hours later stained with
HABC and FITC-streptavidin for newly synthesized, cell-associated hyaluronan.
Examples of areas close to the scraped area (edge) and those more distant
(intact) are shown in mock-transfected (M3), antisense (A21) and sense (S29)
cell lines. Note the higher hyaluronan signal of the wound edge areas in M3
cultures, a low signal in the A21 cells in both intact and edge areas and a
high signal in the S29 cells in both intact and edge areas.
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Fig. 8. Influence of exogenous hyaluronan, hyaluronidase and hyaluronan
oligosaccharides on the migration of the REK cells. (A) 100 µg/ml of
purified hyaluronan was added to the cultures after wounding. The
means±s.e. from nine separate experiments are shown. The asterisk
indicates a statistically significant difference from untreated cultures
(P<0.018, paired t-test). (B) 1 U/ml of
Streptomyces hyaluronidase was added to the culture medium after
wounding. The means±s.e. of three experiments are shown. (C) Hyaluronan
decasaccharides (450 µg/ml) were added to the medium after wounding.
Means±s.e. of six separate experiments are shown.
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© The Company of Biologists Ltd 2002
